neurogenetics (2019) 20:145–154 https://doi.org/10.1007/s10048-019-00581-6 ORIGINAL ARTICLE 9q34.3 microduplications lead to neurodevelopmental disorders through EHMT1 overexpression Maria Teresa Bonati1 & Chiara Castronovo2 & Alessandra Sironi2,3 & Dario Zimbalatti1 & Ilaria Bestetti2,3 & Milena Crippa2,3 & Antonio Novelli4 & Sara Loddo4 & Maria Lisa Dentici5 & Juliet Taylor6 & Françoise Devillard7 & Lidia Larizza2 & Palma Finelli 2,3 Received: 11 April 2019 /Accepted: 28 May 2019 /Published online: 17 June 2019 # Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract Both copy number losses and gains occur within subtelomeric 9q34 region without common breakpoints. The microdeletions cause Kleefstra syndrome (KS), whose responsible gene is EHMT1. A 9q34 duplication syndrome (9q34 DS) had been reported in literature, but it has never been characterized by a detailed molecular analysis of the gene content and endpoints. To the best of our knowledge, we report on the first patient carrying the smallest 9q34.3 duplication containing EHMT1 as the only relevant gene. We compared him with 21 reported patients described here as carrying 9q34.3 duplications encompassing the entire gene and extending within ~ 3 Mb. By surveying the available clinical and molecular cytogenetic data, we were able to discover that similar neurodevelopmental disorders (NDDs) were shared by patient carriers of even very differently sized duplications. Moreover, some facial features of the 9q34 DS were more represented than those of KS. However, an accurate in silico analysis of the genes mapped in all the duplications allowed us to support EHMT1 as being sufficient to cause a NDD phenotype. Wider patient cohorts are needed to ascertain whether the rearrangements have full causative role or simply confer the susceptibility to NDDs and possibly to identify the cognitive and behavioral profile associated with the increased dosage of EHMT1. Keywords 9q34 duplication syndrome . Kleefstra syndrome . EHMT1 . 9q34.3 microduplications . Neurodevelopmental disorders . Autism Introduction interstitial—have been reported to occur without common breakpoints [1]. Chromosome 9q34 subtelomeric region shows genomic insta- Deletions of distal 9q34 encompassing EHMT1 (euchro- bility, as both copy number losses and gains—terminal or matic histone methyltransferase 1) gene (MIM*607001), as Maria Teresa Bonati and Chiara Castronovo made equal contributions and should therefore both be considered as first author Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10048-019-00581-6) contains supplementary material, which is available to authorized users. * Maria Teresa Bonati 4 Laboratory of Medical Genetics, Bambino Gesù Children’s Hospital, [email protected] IRCCS, 00146 Rome, Italy 5 Medical Genetics Unit, Bambino Gesù Children’s Hospital, IRCCS, 1 Istituto Auxologico Italiano, IRCCS, Clinic of Medical Genetics, 00146 Rome, Italy Piazzale Brescia 20, 20149 Milan, Italy 6 Genetic Health Service New Zealand – Northern Hub, 2 Research Lab of Medical Cytogenetics and Molecular Genetics, Auckland, New Zealand Istituto Auxologico Italiano, IRCCS, 20145 Milan, Italy 3 Department of Medical Biotechnology and Translational Medicine, 7 Département de Génétique et Procréation Hôpital Couple-Enfant, Università degli Studi di Milano, Segrate, 20090 Milan, Italy CHU Grenoble Alpes, 38043 Grenoble, France 146 Neurogenetics (2019) 20:145–154 well as loss of function point mutations, are responsible for using the SurePrint G3 Human CGH Microarray Kit 244K in Kleefstra syndrome (KS, MIM #610253), characterized by accordance with the manufacturer’s instructions (Agilent facial characteristics, (childhood) hypotonia, developmental Technologies, Palo Alto, CA). delay (DD), intellectual disability (ID), and other variable Detected copy number variants (CNVs) were classified ac- clinical features [2–4]. Based on multiple reports, 23–100% cording to guidelines reported in Koolen et al. [30], Miller of subjects with KS have autism spectrum disorder (ASD) et al. [31], and Kearney [32]. The following public databases [5–7]. Moreover, a pathogenic variant has been recently iden- were consulted: University of California Santa Cruz (UCSC) tified in a patient with autism and normal intelligence [8]. (http://genome.ucsc.edu/, GRCh37/hg19), Online Mendelian EHMT1 is involved in chromatin remodeling during Inheritance in Man (OMIM) (www.ncbi.nlm.nih.gov/OMIM), neurodevelopment and homeostatic plasticity through synap- ClinVar (www.ncbi.nlm.nih.gov/clinvar/), SFARI, and tic scaling [9]. Database of Genomic Variants (DGV) (http://projects.tcag. A 9q34 duplication syndrome (9q34 DS) has been reported ca/variation/, released in March 2016). A CNV was [10], involving both interstitial [11–14] and terminal [1, 10, classified as rare if unreported or reported at a very low 15–29] duplications, with different sizes. The associated man- frequency (≤ 0.05%) according to the DGV. ifestations include initial poor feeding and thriving, hypotonia, DD, mostly affecting speech and language, ID, craniofacial Fluorescent in situ hybridization analysis dysmorphisms, and other musculoskeletal anomalies. Behavioral problems have been rarely described and include Fluorescent in situ hybridization (FISH) analysis was per- hyperactivity, attention deficit hyperactivity disorder, and formed on P1’s metaphases and interphase nuclei from periph- ASDs [27]. eral blood lymphocytes as previously described [33, 34]. The Starting from the identification of a small microduplication BAC probes RP11-644H13 (Invitrogen Ltd., Carlsbad, CA), encompassing the dosage-sensitive EHMT1 gene in a boy which maps at 9q34.3 and covers EHMT1 from IVS1 to diagnosed in the autism spectrum, our aim was to understand IVS25 (NM_024757), was nick-translation labeled with whether the gene over-dosage may cause a recognizable phe- Cy3-dUTP (Amersham, Chalfont St. Giles, UK). At least 20 notype, and whether this overlaps and/or mirrors that of KS. metaphases and 100 nuclei were screened. Gene expression analysis Materials and methods Total RNA of P1, his father, and 10 healthy controls was Patients collected and isolated using the Tempus Blood RNA tubes and Spin RNA Isolation kit (Thermo Fisher Scientific, Informed consent was obtained from all individual partici- Waltham, MA) and reverse-transcribed with the high- pants included in the study or their legal guardians. Signed capacity cDNA reverse transcription kit (Thermo Fisher informed consent for publication of patient 1’s photographs Scientific). The expression of EHMT1, CACNA1B, SYF2, was obtained from his mother. The study was approved by the and IL1RAPL2 was first verified on a control blood RNA as Ethical Clinical Research Committee of IRCCS Istituto well as on commercial RNAs of human adult and fetal brain Auxologico Italiano. (Clontech Laboratories, Inc., Mountain View, CA) by using Patient 1 (P1) belongs to a cohort of 325 Italian ASD pa- specific pairs of primers. tients that underwent chromosomal microarray analysis RT-qPCR based on the TaqMan methodology was per- (CMA). After his clinical and molecular cytogenetic charac- formed using an ABI PRISM 7900HT Sequence Detection terization, we went on to retrieve detailed clinical and molec- System (Applied Biosystems, Foster City, CA). The amounts ular data of comparable patients reported in public databases, of EHMT1 and SYF2 mRNAs were calculated using the i.e., SFARI (https://gene.sfari.org/) and DECIPHER (http:// 2−ΔΔCt method, with glyceraldehyde 3-phosphate dehydroge- www.sanger.ac.uk/PostGenomics/decipher/). For this nase (GAPDH) and TATA box binding protein (TBP)asthe purpose, we used a uniform questionnaire. By means of this endogenous-normalizing genes. All assays were provided by investigation, further clinical information has been made Thermo Fisher Scientific (TaqMan Gene Expression Assays: available for the two DECIPHER patients referred to as P2 ID# Hs00964325_m1 EHMT1 (ex 14-15, isoforms and P4. NM_024757 and NM_001145527); Hs01548694_m1 SYF2 (ex 6-7, isoform NM_015484.4); Hs99999905_m1 GAPDH; Array CGH analysis Hs00427620_m1 TBP). Real-time data were analyzed using the RQ Manager 1.2 software (Thermo Fisher Scientific). Array comparative genomic hybridization (a-CGH) analysis We established the proper range of gene expression in 10 was performed on genomic blood DNA of P1 and his parents, healthy controls calculating the mean value ± 2 standard Neurogenetics (2019) 20:145–154 147 deviations (SD). If the expression level in the patient was out always consistent with the context. He was toilet trained at of the control range, a dysregulation of the index gene could 44 months; however, anal and bladder sphincter accidents be inferred. could still occur until the age of 7 and 13 years, respectively, when he was tense or not able to ask for the toilet. At the age Gene annotation of 4.9 years, his development was measured by Griffiths scales to be equivalent to that of a 3.8-year-old boy; the total Genes included in the 9q34.3 microduplications were studied IQ was equal to 78 with strengths in the areas of hearing and taking into account their (i) function and possible involvement language and weakness in the performance area. The diagno- in neurodevelopmental disorders (NDDs), according to Gene sis was of pervasive developmental disorder unspecified and Cards (https://www.genecards.org), OMIM, and SFARI; (ii) motor dyspraxia. As shown in Supplementary Table 1,atthe brain expression;